The present invention pertains to non-polypeptide isoindoline derivatives that decrease the levels of tumor necrosis factor alpha (TNFxcex1) and inhibit phosphodiesterases (PDEs), particularly PDE 4 and PDE 3, and to the treatment of disease states mediated thereby. The compounds inhibit angiogenesis and are useful in the treatment of cancer, inflammatory, and autoimmune diseases. For example, compounds that selectively inhibit PDE 4 are useful in treating inflammation and effecting relaxation of airway smooth muscle with a minimum of unwanted side effects, e.g., cardiovascular or anti-platelet effects. The present invention also relates to methods of treatment and pharmaceutical compositions utilizing such compounds.
Tumor necrosis factor xcex1, or TNFxcex1, is a cytokine which is released primarily by mononuclear phagocytes in response to a number immunostimulators. When administered to animals or humans, it causes inflammation, fever, cardiovascular effects, hemorrhage, coagulation, and acute phase responses similar to those seen during acute infections and shock states. Excessive or unregulated TNFxcex1 production thus has been implicated in a number of disease conditions. These include endotoxemia and/or toxic shock syndrome {Tracey et al., Nature 330, 662-664 (1987) and Hinshaw et al., Circ. Shock 30, 279-292 (1990)}; rheumatoid arthritis, Crohn""s disease, IBD, cachexia {Dezube et al., Lancet, 335 (8690), 662 (1990)} and Adult Respiratory Distress Syndrome where TNFxcex1 concentration in excess of 12,000 pg/mL have been detected in pulmonary aspirates from ARDS patients {Millar et al., Lancet 2(8665), 712-714 (1989)}. Systemic infusion of recombinant TNFxcex1 also resulted in changes typically seen in ARDS {Ferrai-Baliviera et al., Arch. Surg. 124(12), 1400-1405 (1989)}.
TNFxcex1 appears to be involved in bone resorption diseases, including arthritis. When activated, leukocytes will produce bone-resorption, an activity to which the data suggest TNFxcex1 contributes. {Bertolini et al., Nature 319, 516-518 (1986) and Johnson et al., Endocrinology 124(3), 1424-1427 (1989)}. TNFxcex1 also has been shown to stimulate bone resorption and inhibit bone formation in vitro and in vivo through stimulation of osteoblast formation and activation combined with inhibition of osteoblast function. Although TNFxcex1 may be involved in many bone resorption diseases, including arthritis, a most compelling link with disease is the association between production of TNFxcex1 by tumor or host tissues and malignancy associated hypercalcemia {Calci. Tissue Int. (US) 46(Suppl.), S3-10 (1990)}. In Graft versus Host Reaction, increased serum TNFxcex1 levels have been associated with major complication following acute allogenic bone marrow transplants {Holler et al., Blood, 75(4), 1011-1016 (1990)}.
Cerebral malaria is a lethal hyperacute neurological syndrome associated with high blood levels of TNFxcex1 and the most severe complication occurring in malaria patients. Levels of serum TNFxcex1 correlated directly with the severity of disease and the prognosis in patients with acute malaria attacks {Grau et al., N. Engl. J. Med. 320(24), 1586-1591 (1989)}.
Unregulated angiogenesis is pathologic and sustains progression of many neoplastic and non-neoplastic diseases including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., Moses et al., 1991, Biotech. 9:630-634; Folkman et al., 1995, N. Engl. J. Med., 33:1757-1763; Auerbach et al., 1985, J. Microvasc. Res. 29:401-411; Folkman, 1985, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203; Patz, 1982, Am. J. Opthalmol. 94:715-743; Folkman et al., 1983, Science 221:719-725; and Folkman and Klagsbrun, 1987, Science 235:442-447. In addition, maintenance of the avascularity of the cornea, lens, and trabecular meshwork is crucial for vision as well as to cel lular physiology. See, e.g., reviews by Waltman et al., 1978, Am. J. Ophthal. 85:704-710 and Gartner et al., 1978, Surv. Ophthal. 22:291-312.
Angiogenesis thus is encountered in various disease states, tumor metastasis, and abnormal growth by endothelial cells. Pathological states created by unregulated angiogenesis have been grouped together as angiogenic dependent or angiogenic associated diseases. Control of the angiogenic processes could lead to the mitigation of these conditions.
The components of angiogenesis relating to vascular endothelial cell proliferation, migration and invasion, have been found to be regulated in part by polypeptide growth factors. Endothelial cells exposed to a medium containing suitable growth factors can be induced to evoke some or all of the angiogenic responses. Polypeptides with in vitro endothelial growth promoting activity include acidic and basic fibroblast growth factors, transforming growth factors xcex1 and xcex2, platelet-derived endothelial cell growth factor, granulocyte colony-stimulating factor, interleukin-8, hepatocyte growth factor, proliferin, vascular endothelial growth factor and placental growth factor. Folkman et al., 1995, N. Engl. J. Med., 333:1757-1763.
Inhibitory influences predominate in the naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis. Rastinejad et al., 1989, Cell 56:345-355. In those instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail.
Macrophage-induced angiogenesis is known to be mediated by TNFxcex1. Leibovich et al. {Nature, 329, 630-632 (1987)} showed TNFxcex1 induces in vivo capillary blood vessel formation in the rat cornea and the developing chick chorioallantoic membranes at very low doses and suggest TNFxcex1 is a candidate for inducing angiogenesis in inflammation, wound repair, and tumor growth.
TNFxcex1 production also has been independently associated with cancerous conditions, particularly induced tumors {Ching et al., Brit. J. Cancer, (1955) 72, 339-343, and Koch, Progress in Medicinal Chemistry, 22, 166-242 (1985)}. Whether or not involved with TNFxcex1 production, angiogenesis is prominent in solid tumor formation and metastasis and angiogenic factors have been found associated with several solid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma, and osteosarcoma. Tumors in which angiogenesis is important include solid tumors, and benign tumors such as acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas. Independent of its action on TNFxcex1 production, the prevention of angiogenesis could halt the growth of these tumors and the resultant damage to the animal due to the presence of the tumor. Angiogenesis has been associated with blood-born tumors such as leukemias and various acute or chronic neoplastic diseases of the bone marrow. In such conditions, unrestrained proliferation of white blood cells occurs, usually accompanied by anemia, impaired blood clotting, and enlargement of the lymph nodes, liver, and spleen.
Angiogenesis also is involved in tumor metastasis. Thus angiogenesis stimulation occurs in vascularization of the tumor, allowing tumor cells to enter the blood stream and circulate throughout the body. After the tumor cells have left the primary site, and have settled into the secondary, metastasis site, angiogenesis must occur before the new tumor can grow and expand.
All of the various cell types of the body can be transformed into benign or malignant tumor cells. The most frequent tumor site is lung, followed by colorectal, breast, prostate, bladder, pancreas, and then ovary. Other prevalent types of cancer include leukemia, central nervous system cancers, including brain cancer, melanoma, lymphoma, erythroleukemia, uterine cancer, and head and neck cancer.
TNFxcex1 also plays a role in the area of chronic pulmonary inflammatory diseases. The deposition of silica particles leads to silicosis, a disease of progressive respiratory failure caused by a fibrotic reaction. Antibody to TNFxcex1 completely blocked the silica-induced lung fibrosis in mice {Pignet et al., Nature, 344:245-247 (1990)}. High levels of TNFxcex1 production (in the serum and in isolated macrophages) have been demonstrated in animal models of silica and asbestos induced fibrosis {Bissonnette et al., Inflammation 13(3), 329-339 (1989)}. Alveolar macrophages from pulmonary sarcoidosis patients have also been found to spontaneously release massive quantities of TNFxcex1 as compared with macrophages from normal donors {Baughman et al., J. Lab. Clin. Med. 115(I), 36-42 (1990)).
TNFxcex1 is also implicated in the inflammatory response which follows reperfusion, called reperfusion injury, and is a major cause of tissue damage after loss of blood flow {Vedder et al., PNAS 87, 2643-2646 (1990)}. TNFxcex1 also alters the properties of endothelial cells and has various pro-coagulant activities, such as producing an increase in tissue factor pro-coagulant activity and suppression of the anticoagulant protein C pathway as well as down-regulating he expression of thrombomodulin {Sherry et al., J. Cell Biol. 107, 1269-1277 1988)}. TNFxcex1 has pro-inflammatory activities which together with its early production (during the initial stage of an inflammatory event) make it a likely mediator of tissue injury in several important disorders including but not limited to, myocardial infarction, stroke and circulatory shock. Of specific importance may be TNFxcex1-induced expression of adhesion molecules, such as intercellular adhesion molecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) on endothelial cells {Munro et al., Am. J Path. 135(I), 121-132 (1989)}.
TNFxcex1 blockage with monoclonal anti-TNFxcex1 antibodies has been shown to be beneficial in rheumatoid arthritis {Elliot et al., Int. J. Pharmac. 1995 17(2), 141-145} and Crohn""s disease {von Dullemen et al., Gastroenterology, 1995 109(I), 129-135}.
Moreover, it now is known that TNFxcex1 is a potent activator of retrovirus replication including activation of HIV-1. {Duh et al., Proc. Nat. Acad. Sci. 86, 5974-5978 (1989); Poll et al., Proc. Nat. Acad. Sci. 87, 782-785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., J. Immunol. 142, 431-438 (1989); Poll et al., AIDS Res. Hum. Retrovirus, 191-197 (1992)}. AIDS results from the infection of T lymphocytes with Human Immunodeficiency Virus (HIV). At least three types or strains of HIV have been identified; i.e., HIV-1, HIV-2 and HIV-3. As a consequence of HIV infection, T-cell mediated immunity is impaired and infected individuals manifest severe opportunistic infections and/or unusual neoplasms. HIV entry into the T lymphocyte requires T lymphocyte activation. Other viruses, such as HIV-1, HIV-2 infect T lymphocytes after T cell activation and such virus protein expression and/or replication is mediated or maintained by such T cell activation. Once an activated T lymphocyte is infected with HIV, the T lymphocyte must continue to be maintained in an activated state to permit HIV gene expression and/or HIV replication. Cytokines, specifically TNFxcex1, are implicated in activated T-cell mediated HIV protein expression and/or virus replication by playing a role in maintaining T lymphocyte activation. Therefore, interference with cytokine activity such as by prevention or inhibition of cytokine production, notably TNFxcex1, in an HIV-infected individual assists in limiting the maintenance of T lymphocyte caused by HIV infection.
Monocytes, macrophages, and related cells, such as kupffer and glial cells, also have been implicated in maintenance of the HIV infection. These cells, like T cells, are targets for viral replication and the level of viral replication is dependent upon the activation state of the cells. {Rosenberg et al., The Immunopathogenesis of HIV Infection, Advances in Immunology, 57 (1989)}. Cytokines, such as TNFxcex1, have been shown to activate HIV replication in monocytes and/or macrophages {Poli et al., Proc. Natl. Acad. Sci., 87, 782-784 (1990)}; therefore, prevention or inhibition of cytokine production or activity aids in limiting HIV progression for T cells. Additional studies have identified TNFxcex1 as a common factor in the activation of HIV in vitro and has provided a clear mechanism of action via a nuclear regulatory protein found in the cytoplasm of cells (Osbom, et al., PNAS 86 2336-2340). This evidence suggests that a reduction of TNFxcex1 synthesis may have an antiviral effect in HIV infections, by reducing the transcription and thus virus production.
AIDS viral replication of latent HIV in T cell and macrophage lines can be induced by TNFxcex1 {Folks et al., PNAS 86, 2365-2368 (1989)}. A molecular mechanism for the virus inducing activity is suggested by TNFxcex1""s ability to activate a gene regulatory protein (NFxcexaB) found in the cytoplasm of cells, which promotes HIV replication through binding to a viral regulatory gene sequence (LTR) {Osborn et al., PNAS 86, 2336-2340 (1989)}. TNFxcex1 in AIDS associated cachexia is suggested by elevated serum TNFxcex1 and high levels of spontaneous TNFxcex1 production in peripheral blood monocytes from patients {Wright et al., J. Immunol. 141(I), 99-104 (1988)}. TNFxcex1 has been implicated in various roles with other viral infections, such as the cytomegalia virus (CMV), influenza virus, adenovirus, and the herpes family of viruses for similar reasons as those noted.
The nuclear factor xcexaB (NFxcexaB) is a pleiotropic transcriptional activator (Lenardo, et al., Cell 1989, 58, 227-29). NFxcexaB has been implicated as a transcriptional activator in a variety of disease and inflammatory states and is thought to regulate cytokine levels including but not limited to TNFxcex1 and also to be an activator of HIV transcription (Dbaibo, et al., J Biol. Chem. 1993, 17762-66; Duh et al., Proc. Natl. Acad. Sci. 1989, 86, 5974-78; Bachelerie et al., Nature 1991, 350, 709-12; Boswas et al., J Acquired Immune Deficiency Syndrome 1993, 6, 778-786; Suzuki et al., Biochem. And Biophys. Res. Comm. 1993, 193, 277-83; Suzuki et al., Biochem. And Biophys. Res. Comm. 1992, 189, 1709-15; Suzuki et al., Biochem. Mol. Bio. Int. 1993, 31(4), 693-700; Shakhov et al., Proc. Natl. Acad. Sci. USA 1990, 171, 3547; and Staal et al., Proc. Natl. Acad. Sci. USA 1990, 87, 9943-47). Thus, inhibition of NFxcexaB binding can regulate transcription of cytokine gene(s) and through this modulation and other mechanisms be useful in the inhibition of a multitude of disease states. The compounds described herein can inhibit the action of NFxcexaB in the nucleus and thus are useful in the treatment of a variety of diseases including but not limited to rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, cancer, septic shock, sepsis, endotoxic shock, graft versus host disease, wasting, Crohn""s disease, inflammatory bowel disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV, AIDS, and opportunistic infections in AIDS. TNFxcex1 and NFxcexaB levels are influenced by a reciprocal feedback loop. As noted above, the compounds of the present invention affect the levels of both TNFxcex1 and NFxcexaB.
Many cellular functions are mediated by levels of adenosine 3xe2x80x2,5xe2x80x2-cyclic monophosphate (cAMP). Such cellular functions can contribute to inflammatory conditions and diseases including asthma, inflammation, and other conditions (Lowe and Cheng, Drugs of the Future, 17(9), 799-807, 1992). It has been shown that the elevation of cAMP in inflammatory leukocytes inhibits their activation and the subsequent release of inflammatory mediators, including TNFxcex1 and NFxcexaB. Increased levels of cAMP also leads to the relaxation of airway smooth muscle.
The primary cellular mechanism for the inactivation of cAMP is the breakdown of cAMP by a family of isoenzymes referred to as cyclic nucleotide phosphodiesterases (PDE) (Beavo and Reitsnyder, Trends in Pharm., 11, 150-155, 1990). There are seven known members of the family of PDEs. It is recognized, for example, that the inhibition of PDE type IV is particularly effective in both the inhibition of inflammatory mediator release and the relaxation of airway smooth muscle (Verghese, et al., Journal of Pharmacology and Experimental Therapeutics, 272(3), 1313-1320, 1995). Thus, compounds that inhibit PDE IV specifically, would exhibit the desirable inhibition of inflammation and relaxation of airway smooth muscle with a minimum of unwanted side effects, such as cardiovascular or anti-platelet effects. Currently used PDE IV inhibitors lack the selective action at acceptable therapeutic doses. The compounds of the present invention are useful in the inhibition of phosphodiesterases, particularly PDE III and PDE IV, and in the treatment of disease states mediated thereby.
Decreasing TNFxcex1 levels, increasing cAMP levels, and inhibiting PDE IV thus constitute valuable therapeutic strategies for the treatment of many inflammatory, infectious, immunological or malignant diseases. These include but are not restricted to septic shock, sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome, post ischemic reperfusion injury, malaria, mycobacterial infection, meningitis, psoriasis, congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer, autoimmune disease, opportunistic infections in AIDS, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, Crohn""s disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolar injury.
The present invention pertains to compounds of Formula I in which the carbon atom designated * constitutes a center of chirality: 
In Formula I, each of R1 and R2, independently of the other, is alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, cyano, cycloalkoxy of 3 to 18 carbon atoms, cycloalkyl of 3 to 18 carbon atoms, or cycloalkylmethoxy in which cycloalkyl has from 3 to 18 carbon atoms, one of X and Xxe2x80x2 is xe2x95x90Cxe2x95x90O or xe2x95x90SO2 and the other of X and Xxe2x80x2 is a divalent group selected from xe2x95x90Cxe2x95x90O, xe2x95x90CH2, xe2x95x90SO2 or xe2x95x90CH2Cxe2x95x90O,
n has a value of 1, 2, or 3;
R3 is xe2x80x94SO2xe2x80x94Y, xe2x80x94COZ, xe2x80x94CN, or hydroxyalkyl of 1 to 6 carbon atoms in which
Y is alkyl of 1 to 6 carbon atoms, phenyl, or benzyl,
Z is xe2x80x94NR6xe2x80x3R7xe2x80x3, alkyl of 1 to 6 carbon atoms, phenyl, or benzyl,
R6xe2x80x3 is hydrogen, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 18 carbon atoms; phenyl, benzyl, or alkanoyl of 2 to 5 carbon atoms, each of which is unsubstituted or substituted with halo, amino, or alkylamino of 1 to 4 carbon atoms, and
R7xe2x80x3 is hydrogen or alkyl of 1 to 4 carbon atoms,
R4 and R5, when taken together, are xe2x80x94NHxe2x80x94CH2xe2x80x94R8xe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94R8xe2x80x94 or xe2x80x94Nxe2x95x90CHxe2x80x94R8xe2x80x94 in which xe2x80x94R8xe2x80x94 is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CHxe2x95x90Nxe2x80x94, or xe2x80x94Nxe2x95x90CHxe2x80x94.
Alternatively, when taken independently of each other, one of R4 and R5 is hydrogen and the other of R4 and R5 is imidazolyl, pyrrolyl; oxadiazolyl, triazolyl, or 
in which
z is 0 or 1,
R6 when taken independently of R7, is hydrogen; alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 18 carbon atoms, alkanoyl of 2 to 5 carbon atoms, or cycloalkanoyl of 2 to 6 carbon atoms, each of which is unsubstituted or substituted with halo, amino, monoalkylamino or dialkylamino in which each alkyl group contains 1 to 4 carbon atoms; phenyl; benzyl; benzoyl; alkoxycarbonyl of 2 to 5 carbon atoms; N-morpholinocarbonyl; carbamoyl; alkoxyalkylcarbonyl of 2 to 5 carbon atoms; N-substituted carbamoyl in which the substituent is alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 18 carbon atoms, or alkanoyl of 2 to 5 carbon atoms, each of which is unsubstituted or substituted with halo, amino, monoalkylamino or dialkylamino in which each alkyl group contains 1 to 4 carbon atoms; phenyl; benzyl; or methylsulfonyl; and
R7 is hydrogen, alkyl of 1 to 4 carbon atoms, or methylsufonyl, or alkoxyalkylcarbonyl of 2 to 5 carbon atoms.
Prerrably z is not 0 when (i) R3 is xe2x80x94SO2xe2x80x94Yxe2x80x94COZ, or xe2x80x94CN and (ii) R4 or R5 is hydrogen.
When taken together, R6 and R7 can be xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94Nxe2x95x90CHxe2x80x94, or alkylidene of 1 or 2 carbon atoms substituted by amino, alkylamino, or dialkylamino in which each alkyl group has from 1 to 4 carbon atoms.
In addition, one of R4 and R5 is: 
in which each of R6, R7, and z is as just define and the other of R4 and R5 is: 
in which zxe2x80x2 is 0 or 1; R6xe2x80x2 has the same meaning as, but is selected independently of, R6; and R7xe2x80x2 has the same meaning as, but is selected independently of, R7.
The present invention also pertains to the acid addition salts of these isoindoline derivatives which are susceptible of protonation. Such salts include those derived from organic and inorganic acids such as, without limitation, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, acetic acid, tartaric acid, lactic acid, succinic acid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid, salicylic acid, phthalic acid, embonic acid, enanthic acid, and the like.
The compounds preferrably are administered as a substantially chirally pure isomer, (S)- or (R)-, but can also be dministered as a mixture of the (S)-isomer and the (R)-isomer.
The compounds can be prepared through a number of methods. Often it is advantageous to utilized protected groups including but not limited to functional groups convertible to the desired group. For example, the reactions described herein can be performed with intermediates in which either or both of R4 and R5 are nitro groups with the nitro group(s) then being catalytically reduced (hydrogenated) to an amine or diamine, as the case may be. Similarly, one can employ an intermediate in which either or both of R4 and R5 is a cyano group and the final compound can then be reduced to yield the corresponding aminomethyl compound. Likewise, the carbonyl comprised by R3 can be processed in the form of a secondary alcohol which is thereafter is oxidized to the carbonyl compound, utilizing for example pyridinium chlorochromate.
Protecting groups utilized herein denote groups which generally are not found in the final therapeutic compounds but which are intentionally introduced at some stage of the synthesis in order to protect groups which otherwise might be altered in the course of chemical manipulations. Such protecting groups are removed or converted to the desired group at a later stage of the synthesis and compounds bearing such protecting groups thus are of importance primarily as chemical intermediates (although some derivatives also exhibit biological activity). Accordingly the precise structure of the protecting group is not critical. Numerous reactions for the formation and removal of such protecting groups are described in a number of standard works including, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, London and New York, 1973; Greene, Th. W. xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, Wiley, New York, 1981; xe2x80x9cThe Peptidesxe2x80x9d, Vol. I, Schrxc3x6der and Lubke, Academic Press, London and New York, 1965; xe2x80x9cMethoden der organischen Chemiexe2x80x9d, Houben-Weyl, 4th Edition, Vol.15/I, Georg Thieme Verlag, Stuttgart 1974, the disclosures of which are incorporated herein by reference.
An amino group thus can be protected as an amide utilizing an acyl group which is selectively removable under mild conditions, especially formyl, a lower alkanoyl group which is branched in 1- or xcex1 position to the carbonyl group, particularly tertiary alkanoyl such as pivaloyl, or a lower alkanoyl group which is substituted in the position xcex1 to the carbonyl group, as for example trifluoroacetyl.
Should a carboxy group require protection, it can be converted to an ester which is selectively removable under sufficiently mild conditions not to disrupt the desired structure of the molecule, especially a lower alkyl ester of 1 to 12 carbon atoms such as methyl or ethyl and particularly one which is branched at the 1- or xcex1 position such as t-butyl; and such lower alkyl ester substituted in the 1- or 2-position with (i) lower alkoxy, such as for example, methoxymethyl, 1-methoxyethyl, and ethoxymethyl, (ii) lower alkylthio, such as for example methylthiomethyl and 1-ethylthioethyl; (iii) halogen, such as 2,2,2-trichloroethyl, 2-bromoethyl, and 2-iodoethoxycarbonyl; (iv) one or two phenyl groups each of which can be unsubstituted or mono-, di- or tri-substituted with, for example lower alkyl such as tert.-butyl, lower alkoxy such as methoxy, hydroxy, halo such as chloro, and nitro, such as for example, benzyl, 4-nitrobenzyl, di-phenylmethyl, di-(4-methoxyphenyl)methyl; or (v) aroyl, such as phenacyl. A carboxy group also can be protected in the form of an organic silyl group such as trimethylsilylethyl or tri-lower alkylsilyl, as for example tri-methylsilyloxycarbonyl.
Many, but not all, of the compounds described herein proceed through compounds in which either or both of R4 and R5 are amino or a protected amino group. The amino group is then further processed as hereinafter described. One can also employ a starting material in which R4 and/or R5 is an amide; e.g., 4-acetamidophthalic acid or 2-chloroacetamide. The product of the latter reaction then can be allowed to react with sodium azide followed by triphenylphosphine to yield a 2-amino-N-substituted acetamide.
In one embodiment, an anhydride or lactone is allowed to react with an xcex1,3,4-trisubstituted benzylamine: 
In the above, at least one of X and Xxe2x80x2 is xe2x95x90Cxe2x95x90O. One also can employ the diacid, e.g., an R4, R5 disubstituted phthallic acid, and remove the water formed. Activated derivative thereof also can be employed.
The compounds in which X is xe2x95x90CH2 can be prepared from the same trisubstituted benzylamine and a formyl or bromomethyl benzoate derivative: 
Analogously, an R4,R5 benzene ortho dialdehyde can be allowed to react with the above xcex1,3,4-trisubstituted benzylamine in the form of the ammonium chloride salt.
The foregoing reactions also can be performed with compound in which R4 and R5 form a heterocylic ring. For example, using furano[3,4-h]quinoline-1,3-dione in place of phthallic acid anhydride, the corresponding 2-substituted pyrrolino[3,4-h]quinoline-1,3-dione is obtained.
When in formula I R4 and R5 are both amino, the compound can be further reacted. Using dimethylformamide dimethyl acetal, for example, yields a pyrrolino[3,4-e]benzimidazole; i.e., R4 and R5 together are xe2x80x94Nxe2x95x90CHxe2x80x94NHxe2x80x94. The corresponding hydropyrrolino[3,4-e]benzimidazole can be obtained from the diamine and triphosgene whereas if one instead employs the diamine and glyoxal, the product is the corresponding 3-pyrrolino[3,4-f]quinoxaline.
In the case of only one of R4 and R5 in formula I being amine, the same can be reacted with an appropriate acid halide or anhydride to yield the corresponding amide. The same reaction can be conducted using chloroformate to yield the methoxycarboxamide derivative.
If the amide is formed from the amine and chloroacetyl chloride, i.e., producing a chloroacetamide derivative, this can be followed by treatment with ammonia or a primary or secondary amine to yield the corresponding aminoacetamide; e.g., treatment with dimethylamine produces the corresponding dimethylaminoacetamide. A compound in which either or both of R4 and R5 is amino also can be subjected to reductive formylation to form the corresponding N,N-dimethylamino compound.
A compound in which either or both of R4 and R5 is amino also can be reacted with dimethylformamide dimethyl acetal to yield the corresponding 1-aza-2-(dimethylamino)vinyl compound.
Compounds in which one of R4 and R5 is a heterocyclic group can be prepared in number of ways. An isoindoline 4- or 5-carboxylic acid can be reacted with carbonyldiimidazole followed by acetic hydrazide to yield the corresponding 4-(5-methyl-1,3,4-oxadiazol-2-yl)isoindoline or 5-(5-methyl-1,3,4-oxadiazol-2-yl)isoindoline. Alternatively, a mono amine and 2,5-dimethoxy-tetrahydrofuran are allowed to react to yield 4- or 5-pyrrolylisoindoline. Similarly a 4-aminomethyl or 5-aminomethyl (prepared as described above) and dimethoxytetrahydrofuran are allowed to react to yield the corresponding pyrrolylmethyl compound.
A first preferred subgroup are those compounds of Formula I in which R4 and R5 together are xe2x80x94NHxe2x80x94CH2xe2x80x94R8xe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94R8xe2x80x94 or xe2x80x94Nxe2x95x90CHxe2x80x94R8xe2x80x94 in which xe2x80x94R8xe2x80x94 is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CHxe2x95x90Nxe2x80x94, or xe2x80x94Nxe2x95x90CHxe2x80x94. It will be appreciated that each of the chains that is not symmetrical can be arranged in either of two orientations, each of which is within the scope of this invention.
A second preferred subgroup are those compounds of Formula I in which one of R4 and R5 is hydrogen and the other of R4 and R5 is imidazolyl, oxadiazolyl, pyrrolyl, or triazolyl.
A third preferred subgroup are those compounds of Formula I in which one of R4 and R5 is: 
in which z is 0 or 1; R6 when taken independently of R7 is hydrogen, alkyl of 1 to 4 carbon atoms, haloalkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 18 carbon atoms; phenyl, benzyl, alkanoyl of 2 to 5 carbon atoms, haloalkanoyl of 2 to 5 carbon atoms, aminoalkanoyl of 2 to 5 carbon atoms, N-alkylamino-alkanoyl of 2 to 5 carbon atoms, benzoyl, alkoxycarbonyl of 2 to 5 carbon atoms, N-morpholinocarbonyl, carbamoyl, and N-substituted carbamoyl in which the substituent is alkyl of 1 to 4 carbon atoms, haloalkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 18 carbon atoms; aminoalkanoyl of 2 to 5 carbon atoms, N-alkylaminoalkanoyl of 2 to 5 carbon atoms, phenyl, benzyl, or methylsulfonyl; and R7 is hydrogen or alkyl of 1 to 4 carbon atoms, or R6 and R7 taken together are xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94Nxe2x95x90CHxe2x80x94, or alkylidene of 1 or 2 carbon atoms substituted by amino, alkylamino, or dialkylamino in which each alkyl group has from 1 to 4 carbon atoms.
Within this third preferred subgroup, a first further preferred subgroup are compounds in which R6 is hydrogen, alkyl of 1 to 4 carbon atoms, haloalkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 18 carbon atoms; phenyl, or benzyl. A second further preferred subgroup are compounds in which R6 is alkanoyl of 2 to 5 carbon atoms, haloalkanoyl of 2 to 5 carbon atoms, aminoalkanoyl of 2 to 5 carbon atoms, benzoyl, alkoxycarbonyl of 2 to 5 carbon atoms, N-morpholinocarbonyl, carbamoyl, and N-substituted carbamoyl in which the substituent is methyl, ethyl, or trifluoromethyl; and R7 is hydrogen.
A fourth preferred subgroup are those compounds of Formula I in which one of R4 and R5 is: 
and the other of R4 and R5 is 
in which each of z and zxe2x80x2 independently is 0 or 1; R6 has the meaning given above, R6xe2x80x2 has the same meaning as, but is selected independently of, R6; R7 has the meaning given above, and R7xe2x80x2 has the same meaning as, but is selected independently of, R7.
Within this fourth preferred subgroup, a first further preferred subgroup are compounds in which each of R6 and R6xe2x80x2, independently of the other, is hydrogen, alkyl of 1 to 4 carbon atoms, haloalkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 18 carbon atoms; phenyl, or benzyl. A second further preferred subgroup are compounds in which each of R6 and R6xe2x80x2, independently of the other, is alkanoyl of 2 to 5 carbon atoms, haloalkanoyl of 2 to 5 carbon atoms, aminoalkanoyl of 2 to 5 carbon atoms, benzoyl, alkoxycarbonyl of 2 to 5 carbon atoms, N-morpholinocarbonyl, carbamoyl, and N-substituted carbamoyl in which the substituent is methyl, ethyl, or trifluoromethyl; and each of R7 and R7xe2x80x2 is hydrogen.
A third further preferred subgroup are compounds in which one of R6 and R6xe2x80x2 is alkanoyl of 2 to 5 carbon atoms, haloalkanoyl of 2 to 5 carbon atoms, aminoalkanoyl of 2 to 5 carbon atoms, benzoyl, alkoxycarbonyl of 2 to 5 carbon atoms, N-morpholinocarbonyl, carbamoyl, and N-substituted carbamoyl in which the substituent is methyl, ethyl, or trifluoromethyl; and the other of R6 and R6xe2x80x2 is hydrogen, alkyl of 1 to 4 carbon atoms, haloalkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 18 carbon atoms; phenyl, or benzyl; and each of R7 and R7xe2x80x2 is hydrogen.
Additional preferred subgroups for all of the above are compounds in which one of X and Xxe2x80x2 is xe2x95x90Cxe2x95x90O, and the other is xe2x95x90Cxe2x95x90O, xe2x95x90CH2, or xe2x95x90SO2, and compounds in which each of R1 and R2, independently of the other, is methyl, ethyl, n-propyl, i-propyl, methoxy, ethoxy, n-propoxy, i-propoxy, cyclopentoxy, cyclohexoxy, cycloheptoxy, cyclopentyl, cyclohexyl, cycloheptyl, or cyclopropylmethoxy.
The compounds possess a center of chirality and thus can exist as optical isomers. Both the chirally pure (R)- and (S)-isomers as well as mixtures (including but not limited to racemic mixtures) of these isomers, as well as diastereomers when there are two chiral centers, are within the scope of the present invention. Mixtures can be used as such or can be separated into their individual isomers mechanically as by chromatography using a chiral absorbent. Alternatively, the individual isomers can be prepared in chiral form or separated chemically from a mixture by forming salts with a chiral acid, or have such as the individual enantiomers of 10-camphorsulfonic acid, camphoric acid, bromocamphoric acid, methoxyacetic acid, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid, and the like, and then freeing one or both of the resolved bases, optionally repeating the process, so as obtain either or both substantially free of the other; i.e., in a form having an optical purity of  greater than 95%.
Inhibition of PDE III, PDE IV, TNFxcex1 and NFxcexaB by these compounds can be conveniently assayed using methods known in the art, e.g., enzyme immunoassay, radioimmunoassay, immunoelectrophoresis, affinity labeling, etc., of which the following are typical.
PBMC from normal donors are obtained by Ficoll-Hypaque density centrifugation. Cells are cultured in RPMI supplemented with 10% AB+ serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin.
The test compounds are dissolved in dimethylsulfoxide (Sigma Chemical), further dilutions are done in supplemented RPMI. The final dimethylsulfoxide concentration in the presence or absence of drug in the PBMC suspensions is 0.25 wt %. The test compounds are assayed at half-log dilutions starting at 50 mg/mL. The test compounds are added to PBMC (106 cells/mL) in 96 wells plates one hour before the addition of LPS.
PBMC (106 cells/mL) in the presence or absence of test compound are stimulated by treatment with 1 mg/mL of LPS from Salmonella minnesota R595 (List Biological Labs, Campbell, Calif.). Cells are then incubated at 37xc2x0 C. for 18-20 hours. Supernatants are harvested and assayed immediately for TNFxcex1 levels or kept frozen at xe2x88x9270xc2x0 C. (for not more than 4 days) until assayed.
The concentration of TNFxcex1 in the supernatant is determined by human TNFxcex1 ELISA kits (ENDOGEN, Boston, Mass.) according to the manufacturer""s directions.
Phosphodiesterase can be determined in conventional models. For example, using the method of Hill and Mitchell, U937 cells of the human promonocytic cell line are grown to 1xc3x97106 cells /mL and collected by centrifugation. A cell pellet of 1xc3x97109 cells is washed in phosphate buffered saline and then frozen at xe2x88x9270xc2x0 C. for later purification or immediately lysed in cold homogenization buffer (20 mM Tris-HCl, pH 7.1, 3 mM 2-mercaptoethanol, 1 mM magnesium chloride, 0.1 mM ethylene glycol-bis-(xcex2-aminoethyl ether)-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (EGTA), 1 xcexcM phenylmethylsulfonyl fluoride (PMSF), and 1 xcexcg/mL leupeptin). Cells are homogenized with 20 strokes in a Dounce homogenizer and supernatant containing the cytosolic fraction are obtained by centrifugation. The supernatant then is loaded onto a Sephacryl S-200 column equilibrated in homogenization buffer. Phosphodiesterase is eluted in homogenization buffer at a rate of approximately 0.5 mL/min and fractions are assayed for phosphodiesterase activity xe2x88x92/+rolipram. Fractions containing phosphodiesterase activity (rolipram sensitive) are pooled and aliquoted for later use.
The phosphodiesterase assay is carried out in a total volume of 100 xcexcl containing various concentration of test compounds, 50 mM Tris-HCl, pH 7.5, 5 mM magnesium chloride, and 1 xcexcM cAMP of which 1% was 3H cAMP. Reactions are incubated at 30xc2x0 C. for 30 minutes and terminated by boiling for 2 minutes. The amount of phosphodiesterase IV containing extract used for these experiments is predetermined such that reactions are within the linear range and consumed less than 15% of the total substrate. Following termination of reaction, samples are chilled at 4xc2x0 C. and then treated with 10 xcexcl 10 mg/mL snake venom for 15 min at 30xc2x0 C. Unused substrate then is removed by adding 200 xcexcl of a quaternary. ammonium ion exchange resin (AG1-X8, Bio-Rad) for 15 minutes. Samples then are spun at 3000 rpm, 5 min and 50 xcexcl of the aqueous phase are taken for counting. Each data point is carried out in duplicate and activity is expressed as percentage of control. The IC50 of the compound then is determined from dose response curves of a minimum of three independent experiments.
The compounds can be used, under the supervision of qualified professionals, to inhibit the undesirable effects of TNFxcex1, NFxcexaB, and phosphodiesterase. The compounds can be administered orally, rectally, or parenterally, alone or in combination with other therapeutic agents including antibiotics, steroids, etc., to a mammal in need of treatment. Oral dosage forms include tablets, capsules, dragees, and similar shaped, compressed pharmaceutical forms. Isotonic saline solutions containing 20-100 milligrams/milliliter can be used for parenteral administration which includes intramuscular, intrathecal, intravenous and intra-arterial routes of administration. Rectal administration can be effected through the use of suppositories formulated from conventional carriers such as cocoa butter.
Dosage regimens must be titrated to the particular indication, the age, weight, and general physical condition of the patient, and the response desired but generally doses will be from about 1 to about 1000 milligrams/day as needed in single or multiple daily administration. In general, an initial treatment regimen can be copied from that known to be effective in interfering with TNFxcex1 activity for other TNFxcex1 mediated disease states by the compounds of the present invention. Treated individuals will be regularly checked for T cell numbers and T4/T8 ratios and/or measures of viremia such as levels of reverse transcriptase or viral proteins, and/or for progression of cytokine-mediated disease associated problems such as cachexia or muscle degeneration. If no effect is observed following the normal treatment regimen, then the amount of cytokine activity interfering agent administered is increased, e.g., by fifty percent a week.
The compounds of the present invention can also be used topically in the treatment or prophylaxis of topical disease states mediated or exacerbated by excessive TNFxcex1 production, such as viral infections, for example those caused by the herpes viruses or viral conjunctivitis, psoriasis, other skin disorders and diseases, etc.
The compounds can also be used in the veterinary treatment of mammals other than humans in need of prevention or inhibition of TNFxcex1 production. TNFxcex1 mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted above, but in particular viral infections. Examples include feline immunodeficiency virus, equine infectious anaemia virus, caprine arthritis virus, visna virus, and maedi virus, as well as other lentiviruses.
The invention thus includes various methods of treatment including the method of inhibiting PDE IV, the method of reducing or inhibiting undesirable levels of TNFxcex1, method of reducing or inhibiting undesirable levels of matrix metalloproteinases, the method of treating undesirable angiogenesis, the method of treating cancer, the method of treating inflammatory disease, the method of treating autoimmune disease, the method of treating arthritis, the method of treating rheumatoid arthritis, the method of treating inflammatory bowel disease, the method of treating Crohn""s disease, the method of treating aphthous ulcers, the method of treating cachexia, the method of treating graft versus host disease, the method of treating asthma, the method of treating adult respiratory distress syndrome, and the method of treating acquired immune deficiency syndrome, by administering to a mammalan an effective amount of a substantially chirally pure (R)- or (S)-isomer of a compound of Formula I or a mixture of those isomers. While these methods may overlap, they also may differ in terms of method of administration, dose level, dosage regimen (such as single or multiple doses), and concurrently administered therapeutic agents.
The invention also includes pharmaceutical compositions in which (i) a quantity of a substantially chirally pure (R)- or (S)-isomer of a compound of Formula I or a mixture of those isomers, that upon administration in a single or multiple dose regimen is pharmaceutically effective is combined with (ii) a pharmaceutically acceptable carrier.
Pharmaceutical compositions can be typified by oral dosage forms that include tablets, capsules, dragees, and similar shaped, compressed pharmaceutical forms containing from 1 to 100 mg of drug per unit dosage. Mixtures containing from 20 to 100 mg/mL can be formulated for parenteral administration which includes intramuscular, intrathecal, intravenous and intra-arterial routes of administration. Rectal administration can be effected through the use of suppositories formulated from conventional carriers such as cocoa butter.
Pharmaceutical compositions will comprise one or more compounds of the present invention associated with at least one pharmaceutically acceptable carrier, diluent or excipient. In preparing such compositions, the active ingredients are usually mixed with or diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule or sachet. When the excipient serves as a diluent, it may be a solid, semi-solid, or liquid material which acts as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, elixirs, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders. Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidinone polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose, the formulations can additionally include lubricating agents such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxybenzoates, sweetening agents or flavoring agents.
The compositions preferably are formulated in unit dosage form, meaning physically discrete units suitable as a unitary dosage, or a predetermined fraction of a unitary dose to be administered in a single or multiple dosage regimen to human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient. The compositions can be formulated so as to provide an immediate, sustained or delayed release of active ingredient after administration to the patient by employing procedures well known in the art.